Unraveling the Complexity of RNA Polymerase I Biogenesis and Transcription Regulation.

The synthesis of ribosomal RNA (rRNA) by RNA polymerase I (Pol I) is central to ribosome production and protein synthesis, processes vital for cell growth and function. Pol I transcription is tightly regulated to balance cellular demands and environmental signals, and its dysregulation is linked to severe diseases, including cancer and Treacher Collins Syndrome (TCS). Despite its fundamental role, Pol I is the least studied RNA polymerases, leaving key aspects of its regulation, assembly, and functional integration within the cell poorly understood. Addressing these gaps is critical to advancing both fundamental biology and the development of therapeutic interventions. This project will address three critical knowledge gaps related to Pol I biology: (1) What are the molecular mechanisms governing the assembly of the multi-subunit Pol I complex, and how are they distinct from the assembly pathways of Pol II and Pol III? (2) How does the Pol I subunits drive liquid-liquid phase separation (LLPS) to regulate Pol I activity and coordinate rRNA processing? (3) How does the Upstream Activating Factor (UAF) shape the chromatin landscape to promote Pol I transcription while repressing Pol II at the same genomic locus? The long-term goal and overarching vision of this proposal are to uncover the fundamental mechanisms of Pol I biology and to translate these insights into therapeutic strategies for diseases such as cancer and ribosomopathies. This cross-organismal study will leverage both yeast and human systems to provide evolutionary insights while ensuring direct relevance to human health and disease. We will employ a multidisciplinary approach combining molecular genetics, proteomics, structural biology, and advanced imaging. Specific strategies include degron-based depletion and proteomics to map Pol I assembly, in vitro and in vivo characterization of Pol I subunit LLPS properties and their regulation by post-translational modifications, and chromatin profiling techniques such as ChIP-Seq and ATAC-Seq to study UAF-mediated chromatin remodeling. Complementary structural analyses using cross-linking mass spectrometry and cryo-electron microscopy will provide insights into the architecture of Pol I and its associated factors. This work will advance fundamental understanding of Pol I biology and its contributions to human disease, while establishing a foundation for future therapeutic development targeting ribosome biogenesis. Project Number: 1R35GM161910-01 | Fiscal Year: 2026 | NIH Institute/Center: National Institute of General Medical Sciences (NIGMS) | Principal Investigator: Bruce Knutson | Institution: RESEARCH FOUNDATION FOR THE STATE UNIVERSITY OF NEW YORK, THE, ALBANY, NY | Award Amount: $448,250 | Activity Code: R35 | Study Section: Special Emphasis Panel[ZRG1 MGG-D (55)] View on NIH RePORTER: https://reporter.nih.gov/project-details/11260830